Peptidoglycan (PGN) and muramyl dipeptide (MDP)

Peptidoglycan (PGN) is present in the cell wall of most Gram-negative and Gram-positive bacteria, however the amount of PGN differs markedly between the two groups of pathogens.

Gram-positive bacteria have a thick layer of a peptidoglycan, which determines the organism's shape (e.g. bacilli, cocci). There may be up to 40 layers of this polymer, conferring enormous mechanical strength to the bacterial cell wall. In contrast, Gram-negative organisms have only a very thin layer of peptidoglycan immediately outside their cell membrane (about

one twentieth of the thickness of that found in gram-positive organisms), surrounded by a bilayered membrane composed of phospholipids and bacterial lipopolysaccharide. The main role of peptidoglycan is to provide certain rigidity and mechanical strength to the cell wall, and therefore to protect bacteria against osmotic lysis.

1.5.3.1 Structure

Peptidoglycan of all bacterial species is composed of long chains of two alternating sugar residues, N-acetylglucosamine (GlcNAc) and N-acetylmuramic acid (MurNAc), which are highly crosslinked by peptide bridges. The peptide subunits consist of alternating L- and D- amino acids and are connected with the carboxyl groups of MurNAc. Among different bacterial species the structure of sugar chains is highly preserved, whereas the composition of the peptide subunits varies. For example, the peptides in S. aureus contain a sequence L-alanine-D-isoglutamine-L-lysine-D-alanine-D-alanine; additionally, PGN from S. aureus contains pentaglycine bridges. The complete peptidoglycan network is a highly complex and ordered network, the detailed structure of which is still under debate (de Jonge et al. 1992;

Dmitriev et al. 2003; Dmitriev et al. 2004). PGN can be enzymatically cleaved into smaller components by two enzymes – N-acetyl-L-alanine amidase and muramidase. The smallest element formed after PGN degradation, which still preserves biological activity, is muramyl dipeptide (MDP) (Ellouz et al. 1974).

1.5.3.2 Biological effects

During Gram-positive bacterial infections PGN is released, together with LTA, and has been shown to co-stimulate the innate immune system. Over the last 30 years a vast number of cellular activities have been assigned to peptidoglycan. Several studies have demonstrated the role of PGN in initiating host cytokine response associated with sepsis and organ injury. PGN was found to induce TNF-α, IL-1β and IL-6 release in human monocytes, with kinetics similar to that induced by LPS (Verhoef and Kalter 1985; Mattsson et al. 1993; Timmerman et al. 1993; Mattsson et al. 1996). Also in a human whole blood model PGN induced the release of TNF-α, IL-6 and IL-10, which coincided with the accumulation of mRNAs for these cytokines in both monocytes and T-cells (Wang et al. 2000). PGN is also known to activate the complement system, as well as to induce procoagulant activity (Mattsson et al.

2002; Mattsson et al. 2004).

The ability of PGN alone to induce nitric oxide synthase (iNOS) has not been proven.

However, evidence from in vitro and in vivo studies suggests that PGN can synergise with

LTA in the induction of iNOS and production of nitric oxide (De Kimpe et al. 1995;

Kengatharan et al. 1996; Kengatharan et al. 1998). Similar synergism was also demonstrated in case of PGN and LPS (Flak et al. 2000; Wray et al. 2001). Recently, PGN from Listeria monocytogenes was also shown to induce oxidative stress and the production of superoxide anion in macrophages (Remer et al. 2005)

It has been argued that PGN is not an important initiator of inflammatory responses because the amounts of this cell wall component needed to induce cellular response are typically high (1-10 µg/ml), several log orders higher than the concentration of LPS. One of the explanations is that only a small part of the PGN structure is essential for its pro-inflammatory activities. PGN is insoluble in its native form and must be enzymatically cleaved into smaller components. Indeed, this smallest active part of PGN has been identified to be the muramyl dipeptide (Ellouz et al. 1974). Only recently, the specific intracellular Nod2 protein has been identified as the receptor for MDP and it was suggested that Nod2 is a general sensor of peptidoglycan through the recognition of MDP (Girardin et al. 2003). MDP itself was shown to activate macrophages (Bahr et al. 1987), monocytes (Kalyuzhin et al.

2002) as well as glial cells (Cottagnoud et al. 2003). On the other hand, in recent studies Traub et al. (2004) demonstrated that the pro-inflammatory activity of MDP could be due to its contamination with LPS, as recombinant MDP not contaminated with endotoxin was not able to stimulate whole blood cells and isolated human monocytes alone. However, it still strongly synergised with LPS to induce cytokine production (Cottagnoud et al. 2003; Traub et al. 2004).

The role of PGN and MDP in the mechanisms of CNS infections has been scarcely studies so far. Nevertheless, the existing evidence suggests that they may play a role in the initiation of the inflammatory response in CNS. In a rabbit model of meningitis, intrathecal injection of MDP triggered TNF-α release and subsequent infiltration of leukocytes (Burroughs et al.

1992; Cottagnoud et al. 2003). In glial cells, MDP was shown to induce PGD2 production (Yamamoto et al. 1988) and to potentiate the cytokine (IFN-γ, IL-1β)-induced iNOS activation and NO production in primary rat astrocytes (Trajkovic et al. 2000).

In the present study we investigated the role of MDP in glia activation. Especially, we were interested whether MDP could act in synergy with LTA in glia and what would be the intra- and extracellular mechanisms involved.